Autophagy is a tightly regulated lysosomal degradation process in which a cell digests its own contents under various forms of duress. In eukaryotic cells, autophagy primarily functions as a protective response during nutrient depletion or stress. Overall, much remains to be learned about how this fundamental process affects cancer initiation, progression and response to therapy. In this proposal, we will test the hypothesis that autophagy influences epithelial cancer development through two mechanisms-1) enhancing the ability of epithelial cells to survive ECM detachment-induced stress and apoptosis (anoikis) and 2) limiting proliferation. This hypothesis is based on initial studies using a unique 3D epithelial culture system to examine how oncogenic insults affect the formation of the normally hollow lumen in glandular structures. In this model, lumen formation involves the selective death of central cells lacking direct contact with extracellular matrix (ECM). Recently, we have discovered high levels of autophagy during lumen formation, indicating that this critical process also contributes to epithelial cell fate. In subsequent work, we are the first to establish that autophagy is strongly induced during ECM deprivation (anoikis). Based on this preliminary data, in Aim 1, we will evaluate whether autophagy promotes cell survival via mitigating the stresses of ECM detachment during anoikis and 3D lumen formation. In addition, we have obtained preliminary evidence that the autophagy regulatory molecule Beclin/ATG6 (a known tumor suppressor) inhibits cell proliferation in 3D culture. In Aim 2. we will interrogate whether other autophagy regulators (called ATGs) inhibit proliferation during growth factor withdrawal and 3D morphogenesis in order to definitively establish that autophagy can restrain epithelial proliferation and to identify new autophagy regulators that are able to inhibit neoplastic cell expansion. In Aim 3. we will delineate how modulating autophagy influences survival and proliferation in oncogene-expressing cells in order to determine how autophagy can be exploited to therapeutically target tumor cells harboring perturbations in known cancer pathways. Overall, the proposed experiments will provide unique information on the precise roles of autophagy during the development and progression of human carcinomas. Ultimately, these studies may lead to new treatment strategies to prevent the uncontrolled expansion of cancer cells via manipulating autophagy.